J 1500 circular dichroism spectrometer

Similar far-UV CD spectra were recorded on an Aviv Model 202 CD Spectrometer for experiments found in Fig. 4 and a JASCO Model J-1500 Circular Dichroism Spectrometer for experiments shown in Fig. S1. Both spectrometers are equipped with a jaacketed cell holder connected to a circulating water bath, and 0.1-cm path length cuvettes were used (Starna). Protein samples were prepared of 80 μM YfiD or 80 μM truncYfiD in 10 mM HEPES, pH 7.2 and 10 mM MES, pH 6.5 (Sigma-Aldrich). The thermal denaturation experiments recorded the CD signal at 220 nm at 5 °C intervals with a 30 sec equilibration time at each temperature range, over the temperature range 20–90 °C. Thermal denaturation curves were obtained by plotting the CD signal at 220 nm as a function of temperature and the melting temperature was determined by fitting the curves to the following two-state model equation:
$$y=\frac{\left[\left(y_f+m_{f}T\right)+\left(y_u+m_{u}T\right)\left(ex{p}^{\mathrm{\Delta }{H}_m/R\left(1/T_m-1/T\right)}\right)\right]}{1+ex{p}^{\mathrm{\Delta }{H}_m/R\left(1/T_m-1/T\right)}}$$
where y is the measured ellipticity, R = 8.3145 J K⁻¹ mol⁻¹, y_f and y_u are the intercepts of the pre- and post-transition baselines, m_f and m_u are the slopes of the pre- and post-transition baselines, T is the temperature (K), ΔH_m is the enthalpy at the unfolding transition and T_m is the melting temperature (K). The equation is fit in R Statistical Software [35 ] using nonlinear least squares to fit all parameters.

UV–vis absorption spectrophotometry was performed using a Jasco V-650 spectrophotometer and 1 cm path length quartz cells. Lyophilized peptide was dissolved in 100 μL of phosphate buffered saline pH 7.4 (PBS; Thermo Fischer), and the concentration of each peptide was determined by measuring the absorbance at 205 nm and using a calculated extinction coefficient for each peptide due to the lack of aromatic residues in the peptides.^{55 (link),56 (link)} For fluorescein-labeled peptides, the lyophilized solid was dissolved in 20 μL of dimethyl sulfoxide (Sigma) and then diluted to 100 μL with PBS. Peptide concentration was determined by measuring the absorbance at 495 nm.
Circular dichroism (CD) experiments were performed using a Jasco J-1500 circular dichroism spectrometer and 1 mm quartz cuvettes. 100 μM samples were prepared for each peptide in sodium phosphate buffer pH 7, and ellipticity was measured from 190 to 260 nm at 5 °C and 95 °C, respectively.
Fourier transform infrared (FT-IR) measurements were collected using a Jasco FT/IR-6800 FT-IR spectrometer. Peptide samples were solvent swapped into deuterated water and deuterated hydrochloric acid. 5 μL droplets of peptide samples were measured at RT, and the absorbance was recorded from 1200–1700 cm⁻¹.